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Abstract

The use of aplanatic solid immersion lenses (ASILs) made of high refractive index optical materials provides a route to wide-field high-resolution optical microscopy. We analyze the aberrations that need to be circumvented for wide-field, high-resolution imaging by hybrid optical simulations based on both ray and wave optics. The conclusions on the relationships among ASIL size, fluorescence imaging spectral width, and field of view not only guide us to design the microscopic system demonstrated in this article, but also clearly demonstrate the general design considerations necessary when applying an ASIL in fluorescence microscopy. Based on the simulation results, we develop wide-field high-resolution solid immersion fluorescence (SIF) microscopy employing an ASIL with an effective numerical aperture (NA) of 1.85. We demonstrate wide-field, high-resolution imaging of synthetic and biological samples with our SIF system. In the presence of a gap between the ASIL and the sample, we experimentally demonstrate that the effective NA of the SIF system is determined by the refractive index of the gap medium in addition to that of the ASIL material; thus, in general, degrading the resolution. Future developments of the SIF system to suit routine use and make it achieve still higher resolution by combining SIF and other microscopic techniques are proposed.

References

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Table 1

Data Depicting the Relationships among ASIL Size, Full Fluorescence Imaging Spectral Width, and FOV When NAeff of the SIF is Maintained at 2 from the SIF Simulationsa

Diameter of ASIL (mm)

Full Fluorescence Imaging Spectral Width (nm)

FOV (μm)

0.5

35

10

1

15

20

3

5

30

5

3

50

8

1

60

a ASIL material, S-LAH79; central wavelength, 632.8nm. The data in bold indicate the parameters adopted in our experiments in which ASIL diameter, full fluorescence imaging spectral width, and FOV were designated as 5mm, 3nm, and 50μm

Table 2

Comparisons of the Refractive Indices of Gap Media, Practical NAs, and NAs Measured from BFPs When Using Different Gap Media in the SIF System

Gap Medium

Refractive Index

Practical NA

NA Measured from BFP

Air

1

1.05

1

Water

1.33

1.28

1.3

Immersion oil

1.518 (at 546nm)

1.47

1.51

High-index liquid

1.78 (at 587nm)

1.7

Not available

Tables (2)

Table 1

Data Depicting the Relationships among ASIL Size, Full Fluorescence Imaging Spectral Width, and FOV When NAeff of the SIF is Maintained at 2 from the SIF Simulationsa

Diameter of ASIL (mm)

Full Fluorescence Imaging Spectral Width (nm)

FOV (μm)

0.5

35

10

1

15

20

3

5

30

5

3

50

8

1

60

a ASIL material, S-LAH79; central wavelength, 632.8nm. The data in bold indicate the parameters adopted in our experiments in which ASIL diameter, full fluorescence imaging spectral width, and FOV were designated as 5mm, 3nm, and 50μm

Table 2

Comparisons of the Refractive Indices of Gap Media, Practical NAs, and NAs Measured from BFPs When Using Different Gap Media in the SIF System